Coxiella burnetiiexploits host cAMP-dependent protein kinase signalling to promote macrophage survival

Abstract

Summary Intracellular bacterial pathogens often subvert apoptosis signaling to regulate survival of their host cell, allowing propagation of the bacterial population. Coxiella burnetii, the intracellular agent of human Q fever, inhibits host cell apoptosis through several mechanisms, including prevention of mitochondrial cytochrome c release, triggering of an anti-apoptotic transcriptional program, and activation of pro-survival kinases. To control host cell survival, C. burnetii delivers effector proteins to the eukaryotic cytosol using a specialized Dot/Icm type IV secretion system (T4SS). Effectors are predicted to regulate activity of pro-survival host signaling proteins, such as Akt and cAMP-dependent protein kinase (PKA), to control infection. Here, we show that host PKA activity is required for C. burnetii inhibition of macrophage apoptosis. PKA is activated during infection and inhibits activity of the pro-apoptotic protein Bad via phosphorylation. Bad is also phosphorylated at an Akt-specific residue, indicating C. burnetii uses two kinases to fully inactivate Bad. Additionally, Bad and the tethering protein 14-3-3β co-localize at the C. burnetii parasitophorous vacuole (PV) membrane during infection, an event predicted to alter Bad promotion of apoptosis. Inhibiting PKA activity prevents Bad recruitment to the PV, but the protein is retained at the membrane during induction of apoptosis. Finally, PKA regulatory subunit I (RI) traffics to the PV membrane in a T4SS-dependent manner, suggesting a C. burnetii effector(s) regulates PKA-dependent activities. This study is the first to demonstrate subversion of host PKA activity by an intracellular bacterial pathogen to prevent apoptosis and survive within macrophages.

Development of an Ex Vivo Tissue Platform To Study the Human Lung Response to Coxiella burnetii

“…Additionally, using a lactate dehydrogenase release assay (Fig. 4B), levels of infected-cell death did not significantly increase compared to those for uninfected cells, similar to the results of our previous apoptosis studies (25)(26)(27). This is unexpected if caspase-1 is activated, as this event typically triggers pyroptosis, an inflammatory form of cell death (23).…”

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“…Nearly all of the agents that target GPCRs are antagonists of signaling receptors for acetylcholine, histamine, serotonin, dopamine, and other adrenergics, suggesting that cellular signaling pathways are required for the intracellular growth of C. burnetii . Utilization of host signaling pathways has previously been observed for several bacterial pathogens, including C. burnetii (12–15). In addition, the proper functions of several host kinases that are downstream effectors of these receptors are essential for the intracellular growth of L. pneumophila , a species closely related to C. burnetii (16).…”

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“…() demonstrated a role for calmodulin kinase II, myosin light chain kinase, protein kinase C and cAMP‐dependent protein kinase (PKA) in CCV development (Hussain et al ., ). PKA regulatory subunit I traffics to the CCV membrane in a manner that is believed to be effector driven (MacDonald et al ., ). Further investigation revealed that PKA activation and the phosphorylation of its downstream targets are differentially regulated throughout the course of Coxiella infection.…”